Extracts from active tree saps for treating P388 mouse leukemia

ABSTRACT

Extracts obtained from boiling displaced tree sap to dryness have significant characteristics especially where solutions are utilized as injectables (e.g., I.P.) in the treatment of P388 mouse leukemia in mice.

United States Patent Hudson Dec. 23, 1975 EXTRACTS FROM ACTIVE TREE SAPSFOR TREATING P388 MOUSE LEUKEMIA [56] References Cited [75] Inventor:Monie S. Hudson, San Jose, Costa OTHER PUBLICATIONS Rlca Hocking, ADictionary of Terms in Pharmacognosy, 73 Assignee: The United States ofAmerica as Charles momas, Springfield, Illinois, pp- 186 represented bythe Department of and 193 Health, Education and Welfare, Abbott et al.,Cancer Research, Vol. 26, part 2, Apr. Washington, DC. 1966, No. 4, pp.391-397, 402 and 534.

22 F1 d: A 5 1972 1 pr Primary Examiner-Jerome D. Goldberg [21 Appl.No.: 241,409

Related U.S. Application Data [57] ABSTRACT [63] Continuation-in-part ofSerUNo. 837,861, June 30, Extracts obtained from boiling displaced treesap to 1969, abafldoneddryness have significant characteristicsespecially where solutions are utilized as injectables (e.g., LP.) inU.S. Cl. the treatmcnt of mouse leukemia in mice [51] Int. Cl. A61K35/78 58 Field of Search 424/195 8 Clam Drawmgs EXTRACTS FROM ACTIVETREE SAPS FOR TREATING P388 MOUSE LEUKEMIA This application is a c.i.p.of Hudson, Monie 8'. Ser. No. 837,861, filed June 30, 1969 and nowabandoned for Novel Composition and Method of Making the Same.

The present invention relates to novel organic compositions derived fromnatural products, or specifically tree saps. The general subject ofnatural products and compounds is of current scientific interest (e.g.,confer CEN, Feb. 28, 1972, page 58, re maytansine and Dr. S. M. Kupchanof the University of Virginia). More particularly, this inventionrelates to dried extracts of specified tree saps which are furtherreconstituted in organic or aqueous media and have been utilized asantileukemia agents against mice afflicted with P388 mouse leukemia. Ithas been further found that only -l0% of several hundred tree sapstested are active against P388 leukemia in mice.

Basically, then, this invention contemplates within its scope, novelcompositions which are comprised of an effective dosage of a solidextract obtained from reducing liquid sap of plant life to a solid.

Generally speaking, the sap of any known plant life is contemplated foruse herein. Such use, of course, is entirely consistent with theabove-related theory that plant growth regulators, etc., found in thesap extract of any regulated plant life also serve as anticancercompounds in animal life. Preferably, however, the sap used is that ofthe various known species of trees.

The sap of trees is particularly preferred for use in this invention formany reasons. For example, the extracts obtained from tree sap have beenfound to have very high anticancer characteristics. Furthermore, thereenters in the practical consideration that only a very small amount ofsolid extract can be obtained from large volumes of liquid sap. Due torecent developments in the timber treatingindustry, wood or tree sap isnow available at relatively low cost in large quantities. Still furtheris the practical consideration that trees are widely available whileother plant life would probably have to be specially farmed over such avast expanse of land to obtain sufficient sap so as to make their useimpractical.

Furthermore, the investigation as here of the activity of dried treesaps appears to be a novel approach. For example, in a 1966 two-partpublication emanating from the Cancer Chemotherapy National ServiceCenter (C.C.N.S.C.-NIH) as follows:

Abbott et al., CR 26, Part 2, pages 39l398, 401,

402, and 534 (1966) Abbott et al, CR 26, Part 2, pages 34, 41, 44, 45,and

1966) there are enumerated criteria for testing plants, bark, leaves.seeds, twigs, etc., particularly at page 40] above and similarcodification at page 44. The investigation into the so-calledreconstituted extracts of previously dried saps as in the presentinvention was a development and a scientific investigation subsequent tothis 1966 protocol.

Examples of various types of trees from which sap may be extractedinclude:

TABLE I Kind of Tree Botanical Name Ailanthus Ailanthus Altissma Alder,smooth Alnus Rugosa Ash, Red Fraxinus Pennsylvanica Green var.Lanceolata Prickly Xanthoxylon Clava-Hcreulis Aspen, Trembling Basswood,white Beech, American Blue Beech Birch, Black River White Yellow Boxelder Buckeye, yellow Butternut Catalpa, southern Cedar, eastern rednorthern white Cherry, black pin Chestnut, American ChinaherryCottonwood, eastern Crabapple, flowering southern Crepe Myrtle Cucumbertree Cypress. bald Arizona Dogwood Elm, American Slippery Winged Fir,fraser Balsam Gum, black sweet Swamp Tupelo Water Hackherry HawthorneHemlock, Eastern Carolina Hickory, Mockernut Pignut Shaghark HopHornbeam Holly, American Mountain Laurel, Mountain Locust, Black HoneyMagnolia, Fraser Southern Magnolia, Sweetbay Maple, Mountain Red StripedSugar Mountain Ash Mulberry, Red Myrtle. Wax

Saltwater Oak, Blackjack Cherryhark Chestnut Live Eastern Red ScarletWater White Willow Paulownia. Royal Persia, Redbay Persimmon Pine, JackLoblolly Longleal' Pond Shortleaf Spruce Slash Tahlcmountain VirginiaWhite Plum. Chickasaw Popular, ,Yellow Carolina Privet RedhudRhododendron, Roseha Populus Tremuloides Tilia Heterophylla FagusGrandifolia Carpinus Carolininiana Betula Lenta Nigra Papyrifera LuteaAcer Negundo Aesculus Octandra Juglans Cinerea Catalpa BignorioidesJuniperus Virginiana Thuja Occidentalis Prunus Serotina PcnnsylvanicaCastanea Dentata Melia Azcdarach Populus deltoidcs Malus Baccata 5 MalusAngustifolia Lagerstroemialndica Magnolia Acuminata Taxodium DistichumCupressus Arizonica Cornus Florida Ulmus Americana Fulva Alata AbiesFraseri Nyssa Sylvatica Liquidamher Stryaciflua Nyssa Billora AquaticaCeltis Occidentalis Crataegus, SSP Tsuga Caroliniana Carya TomentosaOlabra Ovata Ostrya Virginiana llex Opaca Monticola Kalmia LatifoliaRobinia Pseudoacacia Gleditsiu Tricanthos Magnolia Fraseri GrandifloraMagnolia Virginiana Acer, Pennsylvanicum Ruhra.

" Spicatum Saccharum Sorbus Americana Morus Rubra Myrica Cerifera iHeterophylla Ouercus Murilandica Falcata Montana Virginiana RubraCoccinea Nigra Alba Phellos Paulownia Tomentosa Persea BorhoniaDiospyros Virginiana Pinus Banksiana Paeda Palustris var. SerotinaEchinata Glahra Carihea Pungens Virginiana Strohus Prunus AngustiloliaLiriodendron Tulopil'era Populus Canadensia var. Eugenia LigustrumVulgare Cercis Canadensis Rhodendcndron Maximum TABLE l-continuedFurther examples of tree saps, and ones found particularly useful inthis invention because of their unexpectedly high antileukemiccharacteristics, include:

C(NSC Species B 66805) Quercus stcllata B 668100 Salix longipcs B 668090Halesia carolina var. monticola The sap from the plant life used in theinvention may be obtained by any conventional method such as bycrushing, bleeding, and the like. In those instances where tree sap isto be obtained, any one of a variety of methods for extracting the saptherefrom may be used.

Among the conventional techniques currently used for extracting sap fromtrees are the known bleeding (tapping) methods used by the maple syrupindustry and a technique primarily concerned with preserving wood (logs)known as the Boucherie process after its founder.

Since for the input to the present invention tree sap in quantity isnecessary, preferred methods of obtaining the sap usually proceed fromfelled timber where liquids under pressure force the sap from the tree.The basic Boucherie process, described in U.S. Pat. No. 111,784 Smithand US. Pat. No. 2,271,212 Tenger, was designed to replace the sap by awood preservative but is best utilized for the present invention byusing a non-contaminating liquid such as water or acetone.

Recently, improvements in the Boucherie" process have appeared inpatents and in a journal article as follows:

US. Pat. No. 3,427,186 Hudson US. Pat. No. 3,443,881 Hudson Hudson et211., PP] 19, 25, No. 5, May 1969 The improved techniques of US. Pat.No. 3,443,881 above, which use the end cap technique of Boucheriemodified by removing at least one disc cut from the butt endintermediate in the process and the application of sequential liquidpressure treatment, are incorporated by reference in thisapplicationfThis process is also known as the PRESCAP process and apreferred modification for the present invention is set out below.

As adapted for use in this invention, the PRESCAP technique preferablyuses instead of a wood preservative as the first and/or second liquid, aharmless liquid of the same type used in the Boucherie process asadapted for use in this invention. Each liquid, such as 4 water,acetone, or the like, may be the same or a different liquid for the twoseparate steps.

For example, one end (e.g., 6 to 10 inches) of a log from which sap isto be extracted, is debarked, placed and sealed in the above-describedcap in a manner as described in the cited application. The first liquid,such as water, is then forced into the capped end of the log.

The time of treatment depends upon the liquid pressure used. Forexample, a liquid pressure of 200 psi applied for about 2 /2 minutes isvery effective.

In those instances where disc removal is desirable, treatment with thefirst liquid is then stopped and the log is removed from the capwhereinafter a thin disc of wood (e.g., between A; 1 inch in thickness)is removed from the treated end of the log. The log is again resealed inthe cap and a second treating liquid, e.g., water, acetone, or the like,is forced through the log to displace sap from the opposite end thereof.The sap is then collected and treated as hereinafter described.

In a particularly preferred technique of collecting sap, optimum effectsmay be achieved by first extract-- ing sap with acetone and thenentirely re-extract the same log using water. In this respect, theentire procedure as described above is run using acetone as both thefirst and second liquid and then using water as first and second liquid.In so doing, it is found that the acetone treatment will substantiallyincrease the water extractive yield.

It is understood, of course, that extraction of sap is continued untilthe liquid issuing from the collecting end of the log no longer containsa practical amount of sap. Such a point is easily determined by theskilled artisan using conventional techniques.

An additional preferred process is set out in US. Pat. No. 3,427,186Hudson, supra, which process is also known as the SLURRY-SEAL processwhere a slurry of solid particles is utilized to blanket and form a sealover a length of wood for subsequent impregnation or liquid pressuretreatment. The specific techniques uti lized as taught in US. Pat. No.3,427,186 are also incorporated by reference here.

After obtaining the raw sap product by use of the above-describedtechniques, the extract of this invention may be obtained therefrom.Most conveniently,

the extract is obtained by boiling the collected sap to dryness usingknown apparatus such as large pyroceram boiling kettles or the like. Inthis respect, it has been found that for optimum results the sap shouldbe collected and the crude extract obtained therefrom as soon after thetree is cut as possible. This is because biological processes set inrapidly after a tree is cut, to change the chemistry of the wood and thecompounds therein. Generally speaking, the sap should be collected andthe extract obtained within about 30, and preferably 15, days from thetime the trees are initially cut. Such a time period, of course, is onlygeneral, since optimum results vary as the type of tree, time of theyear, etc., is varied.

Large quantities of raw sap are usually required to obtain a usableamount of the extract of this invention.

The actual amount of sap needed will, of course, vary with the type ofWood and the time of the year. As alluded to hereinbefore, optimumyields are usually obtained from many trees just prior to theirproduction of new growth (e.g., just prior to budding in the spring orlate winter). In Northern America, such times usually occur aboutJanuary through March, and even December has been found to be anexcellent month.

Exemplary of the yields to.be expected, in late March, 4 gallons of sapcollected from each of Black gum, Sweet gum, Sourwood, Dogwood, andBlack cherry trees yielded a dry solid extract as contemplated by thisinvention of from about 30 to '50 grams. The

same amount of sap from these species collected in late include southernmagnolia, holly, persea, and the like.

As can be seen from the above, even though some yields are referred toas high, they are, indeed, relatively low with respect to manycommercial chemical processes. It is, therefore, apparent why the use offast sap collecting techniques are most desirable.

As alluded to hereinabove, the solid extract, once obtained, may be usedin its raw form or as a reconstituted extract prepared from the dried orraw sap. For this reason, this invention contemplates within its scope anovel compound comprised, of an effective amount of plant life sapextract as described.

The extract in either its crude or purified form is administeredpreferably l.P., which is the preferred modus in leukemia therapy. Forexample, the extract may be first dissolved in a solvent such as waterand utilized LP. in the bloodstream. In those instances where leukemiais to be treated, the extract may be dissolved in water and injectedintraperitoneally until the malignancy is brought under control. 7

EXAMPLE 1 Extraction and Collection of Sap and Extract from QuercusStellata (Post Oak) A specimen of Post Oak (Quercus Stellata) was cut inJanuary from the Santee Experimental Forest in South Carolina.

The'dimensions were:

Butt dia. in. 4.2 Top dia. in. 3.7 Length, Ft. 7.0

Within 5 days a disc about 1 inch thick was cut from the butt and a capas disclosed in aforesaid U.S. Pat. No. 3,443,881 fitted on the post. Asdescribed above, water pressure at 200 psi was applied to the cap whichforced 4 gallons of sap out the other end during a period of 55 minutes.The rate of flow of sap was 60 gals/sq.ft. of butt area/hr.

The sap was collected and evaporated in a large pyroceram evaporatingdish to dryness. The dried extract weighed 12.0 grams.

EXAMPLE 2 Extraction and Collection of Sap and Extract from HalesiaCarolina var. Monticola (Silverbell) A specimen of Silverbell (HalesiaCarolina var. Monticola) was cut in the vicinity of Murphy, NC, inFebruary. The dimensions were:

Butt dia. in. 4.2 Top dia. in. 3.7 length, Ft. 6.)

The post was prepared for displacement of sap within 2 days after it wascut by cutting off a l-inch disc from the buttand fitting that end ofthe post with the cap of Example 1. Water pressure at 200 psi wasapplied to the cap to displace the sap which was collected. Thefollowing were the observations made:

Rate of Flow Time, Min.

at 200 psi gal.sq.ft/hr.

First Pressure period 99 Second pressure period after 2nd disc cut-otf20 180 Total gallons sap recovered 4.0 Dry extract obtained on Ievaporation 7.2 grams EXAMPLE 3 Dimensions of post:

Top, dia. in. 3.6 Length, Ft. 7.0

Time at Rate of Flow 200 psi, min. gals/sq.ft./hr.

Pressure period 10 482 Total gallons of sap recovercd 4.0 Dry extractobtained by evaporation 6.9 grams EXAMPLE 4 Each of the extractsobtained from Examples l-3 and 14 additional extracts were tested fortheir anticancer effect using the Standard Leukemia Test as reported inCancer Chemotherapy Reports, No. 25, Dept. of Health, Education, andWelfare, December 1962, pp. 1-66. The disclosure of this report isincorporated herein by reference. Basically, the procedure used is asfollows:

Healthy mice, about 3 months old, and weighing about 20 grams (0.7ounces) were injected with a virulent strain of mouse leukemiadesignated P-388. For each test of an extract, 12 mice were used. Six ofthe mice were left untreated after contracting leukemia (i.e., afterbeing injected with P388), while the remaining six mice were injectedintraperitoneally once daily for 10 days using the indicated dosage ofextract dissolved in water.

Survival time of each of the 12 mice was then recorded. The survivaltime of the untreated or control mice was designated as C, while thesurvival time of treated mice was designated as T.

The protocol for determining activity is as follows. The extract isgiven a preliminary approval of Code 1 l or Code 13, where a MC, valueof or better indicating 125% longevity of the treated animal over thecontrol. If satisfactory activity is obtained directly, a Code ll ratingis achieved. If, however, the dosage must be modified for toxicityreasons to obtain the necessary activity, then a Code 13 rating is giventhe extract or compound. Where a second test indicates such a T /C valuewhere the product T,/C -T /C 2 156, the extract is given a Code ratinghaving passed the biological animal (mouse) screen and in the protocolor hierarchy of the C.C.N.S.C. is now a candidate for clinical trial. Inthe above formula, the values for T and C indicate meantreatment/control time for the mouse.

The results below indicate that each of the tree sap extracts possessedactivity at least at the level of Code 15 as read from Screening DataSummary. C.C.N.S.C. (No. I4 of December 1970), which is incorporated byreference to this invention.

Once a Code 15 rating is achieved, additional toxicity testings varyingthe dosage on the same sample (B001) and on a separate sample (B002) areperformed to assist in clinical selection studies.

Of the preferred tree saps, the following were prepared in quantity ofabout 800-1600 gallons and showed Code 15 and 8001 and B002 dosageactivity in the range 100-400 mg/kg per diem:

B 668059 Quercus stellata B 668090 Halesia carolina var. monticola B668100 Salix longipes The following table illustrates the results ofthis testing which was conducted by the Cancer Chemotherapy NationalService Center for Natural Products, National Cancer Institute, ofBethesda, Maryland.

sap is Quercus stellata.

3. The method according to claim 1 wherein the tree sap is Halesiacarolina var. monticola.

4. The method according to claim 1 wherein the tree sap is Salixlongipes.

5. A composition for the treatment of P388 mouse leukemia whichcomprises a pharmaceutically acceptable carrier and an effective amountfor treating P388 mouse leukemia of a tree sap which been producedbyextract with water under 200 psi and subsequent drying and wherein thetree is selected from the group consisting of Quercus stellata, Halesiacarolina var. monticola, and Salix longipes.

6. The tree sap according to claim 5 wherein the sap is Quercusstellata.

7. The tree sap according to claim 5 wherein the sap is Halesia carolinavar. monticola.

8. The tree sap according to claim 5 wherein the tree sap is Salixlongipes.

TABLE ll Screening Results of Plant Saps Active in Leukemia P388 HighestT/C NSC No. Name Locality Code l5. T/C dose BO0I/2 dose 8002/3 dose B66805) Quercus stellata S.C. l55,l 27 400 I 265 I36 265 090 Halesiacarolina var. monticola N.C. I;I30 400 I40 I75 I36 400 I00 Salixlongipes S.C. 135;]30 400 I35 400 I31 I00 Active also at I75 Active alsoat I00

1. A METHOD OF TREATING P388 LEUKEMIA IN MICE WHICH CONSISTS ININJECTING IN SAID MOUSE AN EFFECTIVE AMOUNT FOR TREATING P388 MOUSELEUKEMIA OF A TREE SAP WHICH HAS BEEN EXTRACTED WITH WATER ANDSUBSEQUENTLY DRIED AND WHICH TREE IS SELECTED FROM THE GROUP CONSISTINGOF: QUERCUS STELLATA, HALESIA CAROLINA VAR. MONTICOLA AND SALIXLONGPIPES.
 2. The method according to claim 1 wherein the tree sap isQuercus stellata.
 3. The method according to claim 1 wherein the treesap is Halesia carolina var. monticola.
 4. The method according to claim1 wherein the tree sap is Salix longipes.
 5. A composition for thetreatment of P388 mouse leukemia which comprises a pharmaceuticallyacceptable carrier and an effective amount for treating P388 mouseleukemia of a tree sap which been produced by extract with water under200 psi and subsequent drying and wherein the tree is selected from thegroup consisting of Quercus stellata, Halesia carolina var. monticola,and Salix longipes.
 6. The tree sap according to claim 5 wherein the sapis Quercus stellata.
 7. The tree sap according to claim 5 wherein thesap is Halesia carolina var. monticola.
 8. The tree sap according toclaim 5 wherein the tree sap is Salix longipes.